'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells

In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action

Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase that has an important role in regulating the degradation of cell surface receptors. In the present study we have examined the role of c-Cbl in whole-body energy homeostasis. c-Cb^-/- mice exhibited a profound increase in whole-body energy expenditure as determined by increased core temperature and whole-body oxygen consumption. As a consequence, these mice displayed a decrease in adiposity, primarily due to a reduction in cell size despite an increase in food intake. These changes were accompanied by a significant
increase in activity (2- to 3-fold). In addition, cc-Cb-/- mice displayed a marked improvement in whole-body insulin action, primarily due to changes in muscle metabolism. We observed increased protein levels of the insulin receptor (4-fold) and uncoupling protein-3 (2-fold) in skeletal muscle and a significant increase in the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase. These fmdings suggest that c-Cbl plays an integral role in whole-body fuel homeostasis by regulating whole-body energy expenditure and insulin action.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract

CPE is an aqueous extract of the edible micro alga Chlorella pyrenoidosa, which has been shown to have immunostimulatory effects in vivo. In the present study, CPE was evaluated for an ability to stimulate cytokine production by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were treated ex vivo for 24 hours with 1, 10 and 100 μg/mL CPE. This resulted in a marked increase in the level of IL-10, a regulatory cytokine, and strong stimulation of the T-helper-1 (Th1) cell cytokines, IFN-γ and TNF-α. In contrast, stimulation of representative T-helper-2 (Th2) cell cytokines, IL-4 and IL-13, was minor. CPE (1, 10 or 100 μg/mL) did not cause a proliferation of human PBMC suggesting that enhanced secretion of cytokines was not secondary to an increase in cell number. We conclude that CPE stimulation of human PBMC induces a Th1-patterned cytokine response and a strong anti-inflammatory regulatory cytokine response, observations that await confirmation in vivo.

Vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus (Rajiformes; Rhinobatidae)

This study investigated the nature of previous termvasodilator mechanismsnext term in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus. Anatomical techniques found no evidence for an endothelial nitric oxide synthase, but neural nitric oxide synthase was found to be present in the perivascular nerve fibres of the dorsal aorta and other arteries and veins using both NADPH-diaphorase staining and immunohistochemistry with a specific neural NOS antibody. Arteries and veins both contained large nNOS-positive nerve trunks from which smaller nNOS-positive bundles branched and formed a plexus in the vessel wall. Single, varicose nNOS-positive nerve fibres were present in both arteries and veins. Within the large bundles of both arteries and veins, groups of nNOS-positive cell bodies forming microganglia were observed. Double-labelling immunohistochemistry using an antibody to tyrosine hydroxylase showed that nearly all the NOS nerves were not sympathetic. Acetylcholine always caused constriction of isolated rings of the dorsal aorta and the nitric oxide donor, sodium nitroprusside, did not mediate any dilation. Addition of nicotine (3×10−4 M) to preconstricted rings caused a vasodilation that was not affected by the nitric oxide synthase inhibitor, Image -NNA (10−4 M), nor the soluble guanylyl cyclase inhibitor, ODQ (10−5 M). This nicotine-mediated vasodilation was, therefore, not due to the synthesis and release of NO. Disruption of the endothelium significantly reduced or eliminated the nicotine-mediated vasodilation. In addition, indomethacin (10−5 M), an inhibitor of cyclooxygenases, significantly increased the time period to maximal dilation and reduced, but did not completely inhibit the nicotine-mediated vasodilation. These data support the hypothesis that a prostaglandin is released from the vascular endothelium of a batoid ray, as has been described previously in other groups of fishes. The function of the nitrergic innervation of the blood vessels is not known because nitric oxide does not appear to regulate vascular tone.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Corneal staining and cell shedding during the development of solution-induced corneal staining

Purpose. This non-dispensing cross-over study was conducted to determine if lenses presoaked in Opti-Free RepleniSH (OFR) or ReNu MultiPlus (RMP) cause solution-induced corneal staining (SICS) and subsequent cell sloughing before the typical 2 h in vivo examination point.

Methods. Study lenses (PureVision) were worn bilaterally by 13 participants for periods of 15, 30, 60, and 120 min using two different contralateral care regimen pairings. The lens worn on the test eye was soaked overnight in either OFR or RMP and the control eye in Clear Care (CC). After lens removal, corneal staining was rated on a scale of 0 (negligible) to 100 (severe) for four peripheral quadrants and the central region, and the differential global staining score was calculated by subtracting baseline staining scores. Following the staining assessment, corneal cells were collected from the ocular surface using a non-contact irrigation system to determine ocular cell shedding rates.

Results. Differential global staining score with OFR was greater than CC with the differences being statistically significant at 30 and 60 min (p < 0.01). Maximum staining with RMP was significantly greater than OFR and peaked after 60 and 120 min of lens wear (p < 0.01). On average, 710 ± 470 ocular cells were collected after lens wear, with similar shedding seen independent of solution or lens wear duration (p > 0.05).

Conclusions. SICS occurred earlier but to a significantly lower degree when PureVision lenses were presoaked in OFR compared with RMP, while lenses presoaked in CC did not cause SICS. Ocular surface cell shedding after lens removal was not impacted by lens wear durations of <=2 h.

The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5′GCGCGC3′) or an arbitrary (5′ACGCGT3′) palindrome sequence in place of the 39-nucleotide DIS stem-loop (NLCGCGCG and NLACGCGT). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

IMF®-Therapy (Intention controlled Myo-Feedback) — an innovative method in the treatment of peripheral nerve lesions

Physiotherapy is a well established part of the rehabilitation of peripheral nerve paralysis. The aim of this type of treatment is to re-establish arbitrary functions by improving the patients’ active and passive mobility as well as their strength and stamina. IMF®-Therapy (Intention controlled Myo-Feedback) is an innovative method in the treatment of peripheral nerve lesions that goes beyond the purely neuro-scientific framework and also takes into account methods and concepts of the psychology of learning. The essential assumption is that things learnt in the past are firmly established in the long term motor memory and can be reactivated by the patient. From results achieved in 32 patients treated with this therapy it can be concluded that IMF®-Therapy may be a promising additional rehabilitation tool in peripheral nerve lesion.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

Introduction : While consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation. Methods : In a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content. Results : Erythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content. Conclusion : The data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

Engineering a multimodal nerve conduit for repair of injured peripheral nerve

Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-co-glycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair.

Analysis of 177Lu-octreotate therapy-induced DNA damage in peripheral blood lymphocytes of patients with neuroendocrine tumors

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) can lead to cell death, genome instability and carcinogenesis. Immunofluorescence detection of phosphorylated histone variant H2AX (γ-H2AX) is a reliable and sensitive technique to monitor external beam IR-induced DSBs in peripheral blood lymphocytes (PBL). Here, we investigated whether γ-H2AX could be used as an in vivo marker to assess normal tissue toxicity after extended internal irradiation with (177)Lu-DOTA-octreotate peptide receptor radionuclide therapy (LuTate PRRT) of neuroendocrine tumors.